Part:BBa_K3332087
pTrc-2 derivative-RBS-ECFP-terminator
We use pTrc-2 derivative promoter to express ECFP to check the function of pTrc-2 derivative promoter
With this part, the pTrc-2 derivative promoter can be tested by observing the fluorescence intensity/OD.
Usage and Biology
This part can be used to test that if the pTrc-2 derivative promoter can work. The fluorescence intensity can reflect the strength of the pTrc-2 derivative promoter. Fig 1. pTrc-2 derivative_RBS(B0034)_ECFP(E0020)_terminator(B0015)
Characterization
We use pTrc-2 derivative_E0420_pUC57[BBa_K3332087],pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57 and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57[BBa_K3332089] to characterize pTrc-2 promoter and pTrc-2 derivative promoter. The agarose gel electrophoresis images are below:
note: E0420 is equal to B0034_E0020_B0015.
Protocol:
1.Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution.
2.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.
3.Add 4 mL of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
4.Add 100 μL IPTG stock solution into the induction group when OD600 increased to 0.6.
5.Induce for 6 hours and the condition is the same as before.
6.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500 µl sterile PBS to resuspend.
7.Measure the fluorescence intensity(ECFP)and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group. Here is the result: Fig 5. Fluorescence intensity/OD for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form.
The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative_E0420(ECFP) are used as the positive control group while pLtetO-1_LacI_pTrc2_E0420(ECFP) and pLtetO-1_LacI_pTrc2-derivative_E0420(ECFP) are both experimental groups.
We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.
Reference
[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 37
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
abs | |
biology | |
color | Cyan |
direction | Forward |
emission | CFP |
emit | 476 |
excitation | |
excite | 439 |
kegg | |
lum | |
protein | ECFP |
swisspro | |
tag | None |